Molecular surveillance based on anaplasmosis in domestic small ruminants: First report on zoonotic Anaplasma capra and phylogenetic insights from Faisalabad, Pakistan

Anaplasma is an intracellular alphaproteobacteria that infects diverse blood cell types in animal hosts including small ruminants. Epidemiological and risk factors information on zoonotic anaplasmosis with respect to anaplasmosis in sheep and goats are scarce. Therefore, the objective of the current study was to estimate the prevalence, risk factors of anaplasmosis and phylogenetic investigation of A. capra in sheep and goats from Faisalabad district, Pakistan. Briefly, 384 blood samples were randomly collected from sheep and goats of Faisalabad district, Pakistan, during January to May 2022. The samples were processed for the detection of Anaplasma targeting 16S rRNA gene using PCR. The data regarding disease determinants were collected using a predesigned questionnaire. Out of 384 samples, 131 samples were found positive for Anaplasma spp. with a prevalence rate of 34.11%. The results indicated a significantly higher prevalence of anaplasmosis in goats (41.88%) compared to sheep (22.00%). In addition, the chi square indicated that housing type, tick infestation, gender, tick control practices, age, mix farming, and hygiene were significantly associated with the occurrence of disease. The analysis of multivariate logistic regression expressed gender as the significant risk factor (p = 0.0001, OR = 1.757, CI = 1.305–2.366). The acquired sequences revealed four novel isolates of A. capra (Genbank accession numbers ON834323, ON838209, ON838210, and ON838211). The phylogenetic analysis of the 16S rRNA gene of A. capra revealed three distinct clusters with 99–100% homology with other isolates from different countries. Our isolates showed higher similarity with isolates from China (KM206273, KP314237, MT799937), Pakistan (ON238129, ON238130, ON238131), Angola (MT898988), India (MZ558066), Iran (MW692362), and Turkey (MT632469) isolated from human, sheep, ticks, goats, cattle, Gaddi goat, Persian Onager (Equus hemionus onager), and Turkish goats, respectively. In conclusion, A. capra is endemic in Punjab, Pakistan, there is a need to conduct large scale surveillance studies to assess the status of this pathogen at human-animal interface as well as to develop effective preventive and control strategies to reduce the economic losses associated with anaplasmosis in small ruminants.


Introduction
Livestock is the major subsector of agriculture, contributing 62.68% to agriculture and 14.36% to the national gross domestic product (GDP) of Pakistan.Over 8 million village families are engaged in livestock rearing, driving 35-40 percent of their earnings from livestock [1].Pakistan is the third largest goat producing country in the world with 84.7 million heads.Based on sheep production, Pakistan is the 12 th largest country with 32.3 million populations [1,2].In general, tick-borne diseases (TBDs) are the biggest economic threat to livestock production across the world.Different blood pathogens including Anaplasma, Borrelia, Ehrlichia, Babesia, Theileria and Louping ill virus are the most frequent TBDs in ruminants causing considerable economic losses [3][4][5][6][7].
Anaplasmosis is one of the most prevalent vector-borne illness of domestic and wild animals and humans all over the world including Pakistan [8][9][10][11][12].There are seven recognized species in the genus Anaplasma viz. A. marginale, A. centrale, A. bovis, A. ovis, A. platys, A. phagocytophilum and A. caudatum [13].In addition to this, the researchers also proposed additional species of Anaplasma including A. odocoilei, A. capra and Anaplasma sp.'Omatjenne' [14].Of the aforementioned species of Anaplasma, A. ovis, A. capra and A. phagocytophilum are the major species responsible for anaplasmosis in sheep and goats [4,15].
A new zoonotic tick-borne Anaplasma that gained the attention of veterinary and public health researchers provisionally named as A. capra [16].This organism was the first identified in goats in central and northern China [17].Years later, this was detected in human hospitalized patients of Mudanjiang Forestry Central Hospital, Heilongjiang, China.In addition, twenty-eight human cases have been reported in Heilongjiang Province in northeast China.However, this organism is not yet recognized as a separate species.Anaplasma capra is an emerging zoonotic pathogen and phylogenetically distinct from other Anaplasma species [18].The disease caused by this organism is non-specific signs with fever, malaise, headache, rash, eschar, dizziness, and chills in humans [18,19].The disease caused by Anaplasma species exhibit some level of host specificity, but this feature is distorted due to the detection of Anaplasma in various hosts, which further complicates the epidemiology of the disease.A. capra can infect ruminants like sheep, goat, water buffalo [18,20,21]; wild animals like Muntjac (Muntiacus muntjak), Japanese Serow (Capricornis crispus), deer, Korean water deer (Hydropotes inermis argyropus), Roe dear (Capreolus capreolus), Persian onegar (Equus hemionus onager) [22][23][24]; humans [18]; dogs [25]; mouflon sheep-Ovis gmelini [26] and ticks [27,28].
Giemsa-stained blood smears are frequently used to identify Anaplasma infected animals.Although microscopy is affordable and still regarded as the best, yet it is a laborious technique with low accuracy and requires a skilled examiner [55].Various diagnostic molecular techniques have been utilized for the diagnosis of anaplasmosis such as PCR [18], RFLP-PCR [56], Nested PCR [30], RLB [57] and sequencing [30].Globally, different genes have been targeted for molecular characterization of A. capra, such as a major surface protein 4 (msp4) [20], groEL [47], gltA [18,30] and 16S rRNA [18,26].Pakistan's climate, vegetation, and geographic conditions favor the existence and growth of ticks.Defining the regional status of new zoonotic genotypes of tick-borne pathogens is important at disease control standpoint.Epidemiological and risk factors information with respect to zoonotic A. capra in sheep and goats are scarce.There is only one report of A. capra in mouflon (Ovis gmelini) and domestic sheep (Ovis aries) from Pakistan which differ in terms of sample size and study area.Therefore, the present study was aimed to estimate the prevalence, associated determinants on anaplasmosis and phylogenetic investigation of A. capra in sheep and goats from Faisalabad district, Pakistan.

Study design
The current study was carried out in the Faisalabad district, Pakistan, from January 2022 to May 2022.The study was approved as per ethical guidelines of Graduate Studies and Research Board, Directorate of Graduate Studies, The University of Agriculture Faisalabad, Pakistan, vide letter no.DGS/10925-28, dated March 28, 2022.A total of 384 blood samples were collected from small ruminants (234 goats and 150 sheep) after consent from animal owners using a simple random sampling method.A sample size of 384 was calculated assuming 50% prevalence at 95% confidence using the formula as described by Thrusfield [58].

Risk factors estimation
For estimation of epidemiological risk factors, a pretested questionnaire was filled to gather information after consent at the time of sample collection from each animal owner/farm manager regarding the biotic and abiotic risk factors of age (<2 years and >2 years), gender (male and female), species (goat and sheep), housing type (concrete and muddy), tick infestation (yes and no), hygiene at farm (poor and good), feeding pattern (animals at farm with zero grazing and free range grazing), previous tick history (yes and no), tick control practices (yes and no) and mix farming (present and absent) associated with anaplasmosis.The questionnaire having close-ended questions was completed on spot at the time of blood sampling for each animal.

Blood smear examination
A 5-7 ml blood was drawn aseptically from the jugular vein of sheep and goats with the sterile syringe and shifted into ethylene diamine tetra acetic acid (EDTA) coated vacutainers for conventional assay.From each blood sample, a thin blood smear was prepared as described by Atif et al. [59] and the smear was examined at 100X under oil immersion lens and at least 50 fields per slide were examined to declare a sample as positive or negative [60].

DNA extraction
One milliliter of blood was collected aseptically from EDTA coated vacutainers individually for DNA extraction.The samples were moved to Laboratory of Veterinary Preventive Medicine and Public Health, Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad under cold chain.The DNA was extracted according to the manufacturer's instructions using the commercial QIAamp1 DNA Mini Kit (Qiagen, Germany; catalog No. 69504).A total of 200 ul of individual blood samples were used to extract DNA.The extracted DNA was kept at -40˚C until further testing after being measured for purity and concentration using Nano-drop at 260/280 nm for all samples.

PCR amplification and gel electrophoresis
The PCR was performed targeting 16S rRNA gene of Anaplasma using forward primer HER 16SF (5'-GGTACCYACAGAAGAAGTCC-3'), and reverse primers HER 16SR (5'-TAGCAC TCATCGTTTACAGC-3'), as described by Saleem et al. [61].Final reaction volume of PCR was 20 ul and each reaction contained 10ul Vazyme blue premix (catalog No. P222), A 1 μl forward primer, 1 μl reverse primer, 4 ul water, and 4 ul template DNA was used.Positive and negative controls were also used for the validation of each test.Amplicons were subjected to 2% gel electrophoresis by using 1 μl gel red solution.After solidification, the tray along with the gel was transferred to the electrophoresis tank.The 50 bp DNA ladder (Takara) was loaded in the first well and the samples were loaded into the remaining wells.A 5ul PCR amplicon was loaded in each well of the gel tank, connected to the Power Pac (Bio-Rad, USA) and set to 100-110 volts for electrophoresis at 45-60 minutes.The PCR results were observed with a UV illuminator.

Statistical analysis
Chi square test was performed to determine the association of infection with anaplasmosis and disease determinants.Univariable analysis was used to determine the relationship between anaplasmosis and variables of age (<2 years and >2 years), gender (male and female), species (goat and sheep), housing type (Concrete and muddy), tick infestation (yes and no), hygiene at farm (poor and good), feeding pattern (farmed and free range), previous tick history (yes and no), tick control status (yes and no) and mix farming (present and absent).Multivariable analysis was performed for the variables with values p<0.2 to determine risk factors, odds ratio, and confidence intervals for each variable.The p-value less than or equal to 0.05 was considered as significant.The statistical data was analyzed with IBM SPSS Statistics 26.

Epidemiology
All microscopic positive samples were subjected for further confirmation using PCR.The PCR based overall prevalence of anaplasmosis was 34.11% (131/384).The product of 345bp was perceived on the agarose gel using UV gel illuminator.All the microscopy based positive samples were also found positive from PCR.In the present study, anaplasmosis was more common in goats than sheep.Out of 131 positive animals, 98 goats and 33 sheep were positive for anaplasmosis.The inclusive prevalence of caprine and ovine anaplasmosis was 41.88% and 22%; respectively (Table 1).The Chi square analysis (p<0.05) illustrated that the species-wise prevalence of anaplasmosis was statistically significant (X 2 = 59.93, df = 1, P = 0.000).All PCR positive animals were also found positive based on blood smear microscopy and demonstrated intra-erythrocytic inclusion bodies.The results indicated that Anaplasma is more conjoint in adults than young animals.On the basis of age, animals were categorized into two groups (<2 years and >years).Prevalence was greater in <2 years of age (42.31%; 88/208) than >2 years (24.43%;43/176) age groups.A significant association was found among different age groups (X 2 = 40.29,df = 1, p = 0.000).Gender-wise frequency of disease had a significant association (X 2 = 100.10,df = 1, p = 0.000) indicating a higher positivity in females (41.61%; 114/274) than males (15.45%; 17/110), regardless of host type.The animals were screened for tick infestation to assess the role of ticks on disease outcome.The animals infested with ticks had higher disease positivity rates than tick-free animals with significant association (X 2 = 88.99,df = 1, p = 0.000).All hypothesized risk factors were statistically significant (Table 1).Furthermore, when compared to adult, young animals experienced the disease more frequently.According to univariate and multivariate analysis gender of small ruminants was a statistically substantial risk factor.The analysis of univariate (p = 0.02, OR = 3.532, CI = 1.201-10.387)and multivariate (p = 0.000, OR = 1.757,CI = 1.305-2.366)logistic regression expressed gender as the significant risk factor (Table 2).

Sequencing and phylogenetic analysis
The PCR results based on amplification of 16S rRNA gene of Anaplasma had revealed 131 positive animals out of 384 samples.The representative sequences (n = 04) were sent for sequencing and displayed 99-100% identity with the Anaplasma species.The acquired sequences revealed novel isolates of A. capra with Genbank accession numbers ON834323, ON838209, ON838210, and ON838211.The phylogenetic analysis of the 16S rRNA gene of A. capra revealed three distinct clusters with 99-100% similarity with other isolates from different countries.

Discussion
The primary sources of financial losses for resource-limited livestock farmers in impoverished nations are vectors and vector-borne diseases.Small ruminants are the major source of income for marginal and small farming communities that are heavily dependent on the environment.Due to global warming and climate change, ectoparasites are spreading their habitat to newer areas, putting the previously unexposed hosts at risk of various tick-transmitted pathogens.Anaplasmosis is one of the most significant vector-borne illnesses at human-animal interface.Recently, A. capra got attention from veterinary and public health researchers, and detected in Asian and European countries [16,23,[64][65][66].The A. capra is a zoonotic pathogen also detected in tick vectors such as Haemaphysalis longicornis, H. qinghaiensis, Dermacentor abaensis, D. nuttalli and Rhipicephalus microplus [37,38,41].They have been isolated from humans and goats inhabiting forests in China [18,33].Anaplasma capra has been reported from deer, sheep, and goats in France [23,67] and Onagers (Equidae) in Iran [65].In Pakistan, only one study which depicted A. capra in domestic and Mouflon sheep (Ovis gmelini) which differ in terms of small sample size, outcome, and study location [26].In the first surveillance study, we performed molecular detection, estimated risk factors, and conducted phylogenetic analysis in small ruminants from Faisalabad district, Pakistan.
The results of the present investigation were similar to Belkahia et al. [75], where they depicted higher disease occurrence in female than male animals.This determinant was proved as the most significant risk factor based on univariate analysis.These findings were also supported by Atif et al. [12] as they mentioned higher infection in females than males.This result can be justified by the fact that immunosuppression in females during pregnancy or lactation could be responsible for higher disease outcome in female animals.Belkahia et al. [75], who mentioned a higher infection rates in ewes than in rams.Furthermore, it was revealed that young animals were more infected than adults.Age-related findings are incoherent with Atif et al. [12], who indicated that adults had higher infectivity than young animals.Similarly, the occurrence of anaplasmosis was significantly higher in sheep than goats.As anticipated, anaplasmosis risk was greater in tick-infected animals in this research than non-tick infested animals.Rahman et al. [76], supported our findings who reported comparable results in Jamnapari breed of goats from Bangladesh.Additionally, animals having a history of tick illness had higher disease positivity rates than those who had no history of disease.Our findings are corroborated by the research of Farooqi et al. [77], conducted on cattle in Northern, Pakistan.The mixed farming yield higher infectivity for Anaplasma infection.This had been the fact that animals housed in the same facility might get tick-borne diseases from various animal species.
The highest prevalence was observed in free-grazing animals (39.6%), followed by stall-fed animals (36.5%), and the lowest infectivity in semi-grazing animals (23.9%).Chi square test specified a significant linking between grazing pattern and disease outcome in small ruminants, while regression analysis revealed a non-significant effect.In addition, Yan and colleagues depicted that animals are more prone to infection and the occurrence of anaplasmosis was associated with free-grazing [43].The grazing could have augmented the contact between vectors and other animals [78].Previous studies conducted in Pakistan suggested that grazing animals were at higher risk of infection than their non-grazing counterparts [49].
The acquired sequences revealed four novel isolates of A. capra.The phylogenetic analysis of the 16S rRNA gene of A. capra indicated three distinct clusters with 99-100% similarity with other isolates from different countries.Our isolates showed higher homology with isolates from China (KM206273, KP314237, MT799937), Angola (MT898988), Iran (MW692362.1),India (MZ558066) and Portugal (OK091152.1)isolated from human, tick, goat, cattle, Persian onager (Equus hemionus onager), Gaddi goat and Rhipicephalus microplus ticks, respectively.This is the first report of A. capra in goats from Pakistan.To date, the pathogens remained unnoticed and require large scale surveillance of migratory and non-migratory small ruminants to estimate the gravity of the problem with special reference to vector-transmitted hemoparasites.
Current surveillance studies concluded that zoonotic A. capra is prevalent in small ruminants in Faisalabad district, Pakistan.Gender is the major risk factor for A. capra infection.Phylogenetic analysis indicated novel genotypes.Further studies should be conducted on genetic diversity, host specificity, pathogenicity, vectors competence, and transmission attributes of A. capra zoonotic anaplasmosis using specie specific primers (such as gltA and groEL genes) [79,80] for effective prevention and control of emerging zoonotic pathogen.